Team:SDU-Denmark/Tour73
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Future Laboratory Work
After a screen with the nucleotide library and finding a peptide aptamer against a target protein the next step would be to examine the features of that peptide aptamer.
The peptide aptamers affinity and specificity towards its target are two important parameters to know, especially if it is meant for use in diagnostics and treatment.
The affinity is especially important if we should get more than one positive colony in a screen. Several ways escist, to where we can test this, here among competitive Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA wells would be coated with the target protein, to this we will add the peptide aptamer, giving them time to bind. After binding we will wash out any unbound peptide aptamer. A detection molecule that binds to the peptide aptamer though anti-FLAG will be added and later excess detection molecule will be washed out. An enzyme that converts the detections molecule to a color will be added. The color change or the amount of light emitted is proportional to the level of peptide aptamer bound to target. Reference: Technical Guide for ELISA. Available from KPL, Kirkegaard & Perry Laboratories (Link) Accessed 17 September 2015 Thus the amount of peptide aptamer bound to target protein, provides a measurement for the affinity. The ELISA is competitive, thous the amount of target is small compared realavite to that of peptide aptamers, this means the peptide aptamers will have to “compete” for binding the target protein.
Surface Plasmon Resonance (SPR) is a technology designed by Biacore and could be used to investigate the affinity and the specificity of the peptide aptamer. Central for the SPR method is the movable sensor chip. This chip monitors the interaction between the peptide aptamer and its target. The operation of the instrument, the data and the analyze of the data will be handled by a software. The sensor chip consist of a glass surface covered with a thin layer of gold, which forms a good basis to optimize the binding of a variety of molecules. To this chip the peptide aptamer would be bound. The target proteins will pass over the surface of the chip in a continuous, pulse-free and controlled flow, maintaining constant target protein concentration at the sensor chip surface. SPR measures changes in refractive index, thus senses changes in mass in the aqueous layer close to the sensor chip. Reference: An Introduction to BiaCore's SPR Technology. Available from BiaCore.com (Link) Accessed 17 September 2015 Figure 1 illustrates the principle. This method would besides providing quantitative information of the peptide aptamers affinity towards the target, also provides information of its specificity.
Since we have been working with E. coli BTH101, a β-Galactosidase Assay would also be an option for examine the affinity of the peptide aptamer towards the target. The assay exploits β –Galactosidase’s ability to recognize the synthetic compound o-nitrophenyl-β-D-galactoside (ONPG). The enzyme cleaves ONPG to galactose and o-nitrophenol, which has a yellow color. The production of the yellow color can be used to determine the concentration of the enzyme, since when ONPG is in excess to the enzyme, the production of o-nitrophenol is proportional to the concentration of β-Galactosidase. From the assay, the miller unit can be determined. The miller unit for a known interaction in the system, e.g. our control with the Leuzine Zipper, could be used as a stand that peptide aptamers-target interaction could be compared to. Reference: Open WetWare contributors, "Beta-Galactosidase Assay (A better Miller)," Open WetWare27 August 2012, 23:20 UTC. http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) Accessed 17 September 2015.
The peptide aptamer’s specificity could be examined with protein microarray. Many different proteins would be immoblized to the microarry chip, the peptid aptamer taged with flourecent dye though anti-FLAG, would be added. Thus the fewer proteins towards the peptid aptamer can bind, the higher specificity.